Potassium ions (K+) are necessary for the proper functioning of all cell types. Electrochemical K+ gradients across the plasma membrane and membranes of cellular organelles drive K+ fluxes to control a variety of cell functions. It is well known that fluctuations of the extra- and intracellular K+ concentration control muscle contraction, neurotransmitter and hormone release, neuronal excitability, cell volume, cell proliferation, and cell death. Hence, it is not surprising, that an imbalance of the K+ homeostasis has profound implications at both the cellular and organismal level and is associated with a host of pathological conditions including neurological-, cardio-vascular-, renal-, immunological-, muscle-, and metabolic disorders as well as cancer. K+ fluxes and transport across biomembranes are accomplished by numerous selective K+-channels, exchangers and pumps which emerged as promising therapeutic drug targets for the treatment of various diseases. Our present understanding of extra- and intracellular K+ fluctuations is, however, very limited due to the lack of applicable probes to investigate K+ dynamics with high spatial and temporal resolution.
Strikingly, there is emerging evidence that the K+ concentration within cells control key signaling events also independently from its impact on the membrane potential.
In a recent study, increased intracellular K+ levels were shown to augment the activity of the phosphatase PP2A in T-cells (Eil et al., Nature 537, 539-543 (22 Sep. 2016). As a consequence the Akt-mTOR complex is hypo-phosphorylated and the T-cell effector function suppressed. This study unveils how tightly fundamental cell functions are controlled by K+ ions independently of their contribution to the membrane potential. Moreover, the distribution of K+ among cellular organelles and how dynamic and strong inner organelle K+ concentrations might be affected under certain physiological and pathological conditions have so far not been investigated comprehensively. Our knowledge in this regard is very poor mainly due to a lack of suitable methods and tools that allow quantifying K+ fluxes on the level of individual cells and cellular organelles in real-time. Currently, K+ sensitive electrodes are often used to measure extracellular K+ fluctuations and typically require relatively large sample volumes of at least 1 ml. These electrodes are highly selective for K+ but they can hardly be used to detect the spatiotemporal dynamics of K+ fluctuations and intracellular K+ signals. Several small chemical fluorescent K+ sensors have been developed with the aim to either image extracellular K+ fluctuations or changes of K+ within cells. However, these fluorescent ionic indicators have many limitations as they are often less specific for K+, show a low dynamic range, are K+ sensitive in a non-physiological range, difficult to load into cells and cellular organelles, and in some cases difficult to obtain.
Due to these many severe limitations of fluorescent K+ probes, a meaningful quantitative K+ imaging using fluorescence microscopy and/or fluorimeters is virtually impossible.
Ashraf et al. disclose that a potassium binding protein (Kbp) may in vivo act as a cytoplasmic potassium sensor that is required for normal growth of E. coli at high K+ concentrations. (Structure 2016, May 3; 24(5) 741-9).
WO 01/04623 A1 discloses fluorescence-marked cyclic peptolides (depsipeptides) and their use for optically determining the concentration of potassium ions in a sample.
US 2013/244891 A1 discloses a biosensor comprising an activatable acceptor fluorogen linked by a linker to an environment-sensitive donor that interacts with an analyte.
WO 2012/112440 A2 discloses a fluorescent co-polymer that may function as a potassium ion sensor.
US 2003/119195 A1 discloses fluorescent anthrazene-based fluoroionophores as potassium sensors.
In the light of this prior art, there is still a need to provide further K+ sensors. The development of such sensors is challenging since for example the construction of proximity-based probes is challenging. So far, it cannot be reliably predicted whether the steric effects in a genetically encoded sensor due to ligand binding may induce a conformational change that can be detectable, e.g. by fluorescence quenching or Førster Energy Resonance Transfer (FRET). The suitability of a genetically encoded sensor thus depends on a case-by-case basis on the individual binding domain, the extend and stability of its conformational change upon ligand binding and optional linker sequence between the binding module and the detection domains.
Thus, it is an object of the present invention to provide novel agents suitable for the detection of K+.
Further, it is another object of the present invention to provide novel methods for detecting K+ in a sample.